Finish Genetically Modified (GM) Foods

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Oct 22, 2013 (4 years and 18 days ago)

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Finish Genetically Modified (GM) Foods

Yesterday
-

UK approved first GM crop for planting (with strict guidelines


Herbicide
-
tolerant corn


‘8,000,000 farmers in 18 countries are now growing GM crops’

Source: Sci. Am. April 2001

Major GM crops
and how they are
modified

Year 2000

Model organism for tree genomics

Timber, plywood, pulp, paper

Fast growth
-

7 year old poplar
stand in Oregon

Trees too!

Poplars and aspens
-

genus
Populus

Engineering wood (cell wall) for
better pulp quality, etc.

Lecture 13

Molecular Manipulations: Genes, Genomes
and Biotechnology

Genes and Genomics

Biotechnology
-

genetically modified organisms (GMOs)

GMO Overview


*The Science


Herbicide and insect resistant plants


The major concerns


Herbicide use will increase


Gene pollution


Unintended toxicity to animals



Are GE foods safe?


Most common modifications

Herbicide tolerance

-

Roundup
-
ready™ plants, contain
gene that makes plant resistant to herbicides

Insect resistance

-

Bt plants, contain toxin gene from Bacillus
thuringiensis that kills larvae

Glyphosate (Roundup™; Monsanto) blocks shikimate pathway

Shikimate pathway
-

Biosynthesis of aromatic
amino acids (trp, phe, tyr)

Glyphosate binds to and
inhibits EPSP synthase

Not in animals

Glyphosate =

N phosphomethyl glycine


P
-

CH
2

-

NH
-

CH
2

-

COO
-

Some EPSP synthases from
bacteria are resistant to
glyphosate (single aa change
Gly
96

to Ala)

35S promoter

(CMV)

EPSPS(Agrobacterium)


Transform cotton cells in culture, plasmid inserts in
genome


Grow cells in presence of antibiotic


Regenerate plant from transformed cells


Test protein levels and glyphosate resistance

Ti Plasmid

Amp
r

(ampicillin
resistance)

Replication
origin

Multiple
cloning
site

Cotton EPSPS

Agrobacterium EPSPS


Note that plant will have 3 EPSPS

Roundup
-
ready™ cotton, soybeans
-

Monsanto

See ECB 10
-
40

Source Sci. Am. April 2001

Bt corn

‘Plant cells are totipotent’

Transformation
-

some cells will take up
plasmids, others use gene gun (biolistics)

Roundup
-
ready™ soybeans

Untreated
-

weed infested

Sprayed with Roundup™

Clone gene coding for BT toxin
-

pesticide


(several companies)

Protein toxin from
Bacillus thuringiensis


Kills larvae of



Lepidopterans (butterflies, moths)



Dipterans (2 winged flies (gnats, mosquitos))



Coleopterans (beetles)


Agricultural importance
-

Kills corn borer, corn root worm and cotton
bollworm larvae

Insect resistant plants

Corn borer

Corn root worm

Bt Corn from Phillipines


Mechanism of toxin action:


Binds to receptors in insect gut


Ionophore
-

ion channel that allows ions to flow across

plasma membrane

Note: organic farmers spray crops with intact Bt bacterium

Cotton bollworm

Cotton
-

#1 pesticide using crop, a
major pollutant environmentally.


Bt cotton has solved this problem.


But raised others, effects on
butterflies……

Lecture 13

Molecular Manipulations: Genes, Genomes
and Biotechnology

Genes and Genomics

Biotechnology
-

genetically modified organisms (GMOs)

GMO Overview


The Science


Herbicide and insect resistant plants


*The major concerns


Are GE foods safe?


Herbicide use will increase


Gene pollution


Unintended toxicity to animals



Regulatory oversight

Environmental Protection Agency
-


Safe for the environment?

US Department of Agriculture
-

Safe to plant?

Food and Drug Aministration
-


Safe to eat?


A new protein not already in diet must be shown to be safe


GRAS
-

‘generally recognized as safe’. If protein is not significantly different

from one already in diet. (EPSPS, most Bt)


In consultation, plant must look normal, grow normally, taste normal and have

expected levels of nutrients and toxins


In 2001, request data on bioengineered crops 120 days prior to commercial distribution


To date, no evidence that a GM crop is unsafe to eat. Starlink corn….


Source: USDA website

Concern: Are genetically modified foods safe to eat???


Regulatory oversight

Starlink™ corn

In 2000 Starlink™ Bt corn from Aventis was found in Kraft
taco shells

Starlink™ Bt corn had not approved for human consumption

Worse, a watchdog group, not the FDA, found the tainted taco shells

Concern was that Starlink™ Bt corn was an allergen;


but in November 2003, scientists reported that additional


tests had failed to demonstrate the presence of an allergen


in the modified corn

Gene will be introduced into wild populations when
transgenic pollen is
carried to compatible plants





Serious concern for


Cotton and wild relatives

in southern US


Corn and teocinte

in Mexico and Guatemala



Evidence from Mexico that bioengineered gene is in



wild populations


Could result in herbicide resistant weeds and Bt containing wild plants



Possible solutions:

Clone into chloroplast genome

which is inherited maternally in most plants

Male sterile plants


Concern: Introgression (gene pollution)

Glyphosate up; overall use slightly reduced

Source USDA AER 786

Concern: Herbicide use will go up

Concern: Toxicity to unintended animals


Bt is biggest worry


Toxicity of transgenic pollen

Bt pollen may be carried to nearby plants
(milkweed) and eaten by non
-
pest (
monarch
butterfly
)

Risk assessment

Sears et al. (2001) PNAS 98, 11937; “impact of Bt corn pollen from
current commercial hybrids on monarch butterfly populations is
negligible
.”


*

US already overproduces food


Major problem in 3rd world is distribution



*

Resistance to pesticides (Bt) will be selected for


GE crops are only short term solutions


*

Gene may be transmitted from GM field to organic crops


Almost certainly will happen, British very concerned


*

Labeling of foods


Europe and Japan
-

Label and segregate (if EU lifts current ban)


US
-

voluntary, although public supports mandatory labeling


*
Additional oversight; testing and scientific studies


Agencies currently reviewing their policies


*
Enforcement


Starlink™ Bt corn


Other issues

Future directions

2005
-
2015

Resistance to herbicides, pests and pathogens

Tolerance to drought, salt, heavy metals and low/high temperature

Improved nutritional quality (proteins, oils, vitamins, minerals)


Golden rice
-

engineered to synthesize
b
-
carotene,


vitamin A precursor


Vit. A deficiency causes blindness

Improved shelf life of fruits and vegetables

Improved flavors and fragrances

Elimination of allergens

Production of vaccines, human therapeutic proteins,


pharmaceuticals

Phytoremediation

Vasil, Nature Biotechnology 21; 849
-
51 (2003)

Beyond the central dogma

Central dogma culminates with synthesis of protein in
cytoplasm


But can’t mix proteins, polysaccharides, lipids and nucleotides
together and get a living cell


Formation of a cell requires the context of a pre
-
existing cell


Cell structures (organelles; mitochondria, chloroplasts, Golgi,
ER) and organization must be inherited, just like DNA


Epigenetics



Lecture 14
-
17 Endomembrane System


Protein targeting, secretion, and vesicle traffic


Targeting proteins to cytoplasmic organelles


Targeting and translocating proteins into the ER


Vesicle trafficking, budding and fusion (ER, Golgi,lysosome


Endocytosis



Today L14, protein targeting to cytoplasmic organelles




Protein folding and degradation


Intro to targeting


Import into nucleus


Import into chloroplast and mitochondria

Nascent proteins must fold to the
correct II
o

and III
o
conformation

Folding of the nascent polypeptide begins during translation

Information for folding is in amino acid sequence:
fold to minimum
energy configuration

Some proteins can fold (and can refold) spontaneously

Some proteins can’t

From
MBoC

(4) figure 6
-
81 © Garland Publishing

mRNA

N
-
terminal domains fold

C
-
terminal
domains fold

Completed protein
released from ribosome
(a
few minutes after translation
began)

ATP +

+ ADP + Pi

HSP60 family

“Heat shock (HSP)” proteins aid protein folding

HSP70

family ATPases act as “chaperones” to aid protein folding

Adapted from MBoC(4)

figures 6
-
83 and 6
-
84

Correctly
folded protein

Incorrectly
folded protein

ATP

ADP
+ Pi

ADP

ATP hydrolysis

Exchange

HSP70 binds
hydrophobic regions

HSP70 released

Correctly
folded protein

“Proteosome”

HSP60

family of chaperones tries to
re
-
fold mis
-
folded proteins…

ATP

Synthesis of chaperones increases dramatically at elevated T

Death of a protein: mis
-
folded, damaged, or
unneeded proteins are degraded in proteosomes

Cytoplasmic enzymes recognize
mis
-
folded

(up to 1/3 of newly synthesized proteins),
damaged
, or
short
-
lived

proteins

…and “tag” those proteins for destruction
by covalently linking
ubiquitin

(76 aa
polypeptide) to lysine side chains.

Short
-
lived proteins may contain specific
“destruction” sequences that target them
for rapid
ubiquitination


Tagged proteins are then degraded in
“proteosomes.”


ubiquitin (
?
)

ATP

AMP+
2 Pi

Incorrectly folded or
damaged protein

Ubiquitinated protein
marked for degradation

“Proteosome”

Peptides

The proteosome
ECB 7
-
36

20S
proteosome

19S cap = ‘gate’

Active sites

Lecture 14


Protein folding and degradation



Intro to protein import into organelles


Import into the nucleus


Import into mitochondria and chloroplasts

Review: Prokaryotes have few “compartments”

Nucleoid
(packaged DNA)

Cytoplasm

ECB

figure 1
-
11

…in contrast to eukaryotic cells, which have many compartments

Nucleus

(
DNA replication, transcription
and RNA processing
)

ER

(
lipid metabolism; synthesis of
secretory and membrane proteins
)


Golgi

(
processing and sorting of
secretory and membrane proteins
)

Mitochondria and chloroplasts

(
ATP
synthesis and carbon fixation
)

Endosomes

(
endocytosis
)

Lysosomes

(
recycling
)

Peroxisomes

(
detoxification
)

Cytosol

(lots of things)

ECB
panel 1
-
2 and
figure 15
-
2

Relative numbers and volumes of some membrane
-
bounded compartments in a hepatocyte (liver cell)

Qs for next few lectures

How are proteins targeted to the correct compartments?

How do these organelles communicate with each other?

Nucleus

1

6

Sequesters genome.

Mitochondria

1700

22

TCA, resp., ox phos etc



ER

1

12

Lipid synthesis. Synthesis

of secreted and membrane

proteins.

Golgi

1

3

Processing and s

orting

membrane/secreted

proteins.

Peroxisomes

400

1

Oxidative detoxification.

Lysosomes

300

1

Degradation and recycling.

Endosomes

200

1

Sorting.

Cytosol

1

54

Metabolism and protein

synthesis.

Adapted from
ECB

Tables 15
-
1 and 15
-
2

Compartment

Number/cell

Relative volume (%)

Function

Plant cell
-

most of volume is vacuole, dozens to 100s of chloroplast

Origin of nucleus and ER

Invagination of plasma membrane

Nucleus surrounded
by double membrane

Outer nuclear
membrane is
contiguous with ER

ECB 15
-
3

Origin of mitochondria and chloroplasts

ECB 15
-
4

Surrounded by double membrane and contain own DNA, but codes for
very few proteins! (a few dozen)

Instead, most genes from prokaryotic ancestor have been transferred
to the nucleus, so proteins must be imported

Three ways organelles import proteins

15_05_import_proteins.jpg

ECB 15
-
5

We will begin with import
into nucleus and then
consider chloroplast and
mitochondria

Then import into ER and
protein transport to Golgi,
lysosomes etc. via vesicles

Import into organelle from cytoplasm is
directed by sequence in protein


Specific aa sequence for each organelle, often near amino terminus

Typically 15
-
60 aa long, usually removed after import

Often not a specific sequence but hydrophobicity or placement of
charged amino acids

(NLS)

Signal sequence is both necessary and sufficient for
import

Necessary

Sufficient

Study import into organelles using molecular tools

Green fluorescent protein (GFP) is cloned onto protein of interest

GFP


Promoter

Test protein

Construct is transformed into cells where it is transcribed and translated

Virus protein::GFP


virus protein(
-
NLS)::GFP


NLS::GFP

Cell

GFP

merge

Import into nucleus

End L14 2004